DESCRIPTION: Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. The applicant has formulated a working model of the subunit interactions responsible for TH and TPH tetramer formation in which sequence elements in both the N-terminal and C-terminal domains are required for creating mature tetrameric enzymes. Experiments are required to determine the specific residues involved in subunit assembly and enzyme activity as well as to determine the consequence of tetramer formation in a cellular environment. The applicant proposes experiments for both TH and TPH to test the hypothesis that higher order macromolecular structures are required for the allosteric regulation of enzyme activity (aim 1). Studies are proposed that will refine the current understanding of subunit assembly by employing more selective mutagenesis and that will focus on enzyme kinetics and phosphorylation-activation of the enzymes in vitro. In aim 2, the role of higher order enzyme structures in the cellular control of hydroxylase activity will be examined by transferring selected recombinant enzymes into eukaryotic cells. This approach is designed to assess the effects of quaternary structure (ie, monomer vs tetramer) on enzyme stability, as well as on regulation by endogenous second messenger pathways. In addition to providing valuable information concerning the basic biology of hydroxylase function, the principal investigator argues that these experiments will guide future efforts in transgenic animal production. In aim 3, the applicant proposes to extend his recent observations that the related hydroxylase, tyrosinase, acts as a quinone generator to convert dopamine into a covalent suicide inhibitor of TH. This observation not only suggests a new molecular basis for dopamine neurotoxicity, but provides a novel tool for identifying important active site amino acid residues. Utilizing radiolabeled dopamine, modified residues will be identified followed by the selective mutagenesis of these residues in recombinant TH. Given the highly homologous structures of TH and TPH, the findings of these studies with the former enzyme will then be extended to the latter. Given the nature of biogenic amines in human health, the applicant argues that it is important to understand the two primary biosynthetic enzymes. The proposed experiments are designed to extend the applicant's progress in this field and to enhance our knowledge of hydroxylase function.